Understanding the relationship between the linear amino acid sequence of a protein and its three-dimensional structure remains one of the most important goals in biological chemistry. In this research, novel chemical approaches will be used to systematically study the structural stability of the Src Homology 3 (SH3) protein domain family. The long term objectives of this work are to identify the key regions within the chemical structure of an SH3 domain crucial to maintain the native protein fold, both in the presence and absence of bound ligand. Knowledge gained from these investigations will lay a strong foundation for the future de novo design of SH3 domains containing only the minimum sequence determinants for folding. The three specific aims of this proposal share a common objective, which is to acquire a chemical etiology of the tertiary structure of this commonly occurring protein domain. (1) The complete chemical structure of an SH3 domain will be systematically varied using a novel chemical- driven approach which combines the valuable information gained by systematic modification of a protein~s chemical structure, with the synthetic advantages of combinatorial methods. Using functional and structural selection assays, we will evaluate the qualitative effects of scanning a series of chemical modifications through the primary sequences of the SH3 domains from the adaptor protein, c-Crk, and the protein tyrosine kinase, c-Abl. (2) The SH3 domain fold provides an excellent model system for the generation of cyclic proteins. A chemoselective ligation approach will be used to covalently link the N- and C-termini of the SH3 domain from c-Abl. The Structural, thermodynamic and biological properties of the resulting cyclic SH3 domain will be studies in some detail. These studies will provide important insights into how altering the entropy of the system through cyclization, effects the folding and functional properties of the protein. (3) A combinatorial approach will be used to generate every possible circularly permuted form of c-Abl SH3. This chemistry-driven strategy relies on an initial chemoselective ligation event followed by the specific cleavage of a synthetic insert placed at every position within the sequence of the protein. Structural and functional analysis of the resulting mixture of circular permuters will provide information into the role of the N- and C-terminal regions in protein folding.